GST Isoforms Research Articles

The acute effects of microcystin LR on the transcription of nine glutathione S-transferase genes in common carp Cyprinus carpio L.

The glutathione S-transferases play important roles in the detoxification of microcystin. In this experiment, nine glutathione S-transferase genes including cytosolic GSTs (rho, mu, theta, alpha and pi), mitochondrial GST (kappa) and microsomal GSTs (mGST1, mGST2 and mGST3) were cloned from common carp Cyprinus carpio. The mRNA abundance of each carp GST isoform in liver was analyzed by real time PCR. The relative changes after stimulation with microcystin LR were also analyzed: increased levels of transcription of GST alpha, rho and mGST3 isoforms were detected at 6 h post stimulation; the transcription of mu, theta and mGST2 isoforms were relatively stable; and all the GST isoforms except GST kappa and rho recovered to original levels compared with controls at 72 h. It is suggested that MC-LR showed different effects on the transcription of nine carp GST isoforms.

Inhibition of cytosolic glutathione S-transferase activity from rat liver by copper

H(2)O(2) inactivation of particular GST isoforms has been reported, with no information regarding the overall effect of other ROS on cytosolic GST activity. The present work describes the inactivation of total cytosolic GST activity from liver rats by the oxygen radical-generating system Cu(2+)/ascorbate. We have previously shown that this system may change some enzymatic activities of thiol proteins through two mechanisms: ROS-induced oxidation and non-specific Cu(2+) binding to protein thiol groups. In the present study, we show that nanomolar Cu(2+) in the absence of ascorbate did not modify total cytosolic GST activity; the same concentrations of Cu(2+) in the presence of ascorbate, however, inhibited this activity. Micromolar Cu(2+) in either the absence or presence of ascorbate inhibited cytosolic GST activity. Kinetic studies show that GSH but no 1-chloro-2,4-dinitrobenzene prevent the inhibition on cytosolic GST induced by micromolar Cu(2+) either in the absence or presence of ascorbate. On the other hand, NEM and mersalyl acid, both thiol-alkylating agents, inhibited GST activity with differential reactivity in a dose-dependent manner. Taken together, these results suggest that an inhibitory Cu(2+)-binding effect is likely to be negligible on the overall inhibition of cytosolic GST activity observed by the Cu(2+)/ascorbate system. We discuss how modification of GST-thiol groups is related to the inhibition of cytosolic GST activity.

Expression of glutathione S-transferases in fetal lung and liver tissue from parental strains and F1 crosses between C57BL/6 and BALB/c F1 mice following in utero exposure to 3-methylcholanthrene

GST isoforms have been extensively studied in adult tissues but little is known about the composition and levels of these enzymes in fetal tissues. As part of our ongoing studies to determine the potential role of metabolic enzymes in mediating the differential susceptibility of different strains of mice to lung tumorigenesis following in utero exposure to 3-methylcholanthrene (MC), we screened for GST enzyme activity and for expression of the individual GSTα, π, μ, and θ isoforms in murine fetal lung and liver tissues isolated from the parental strains and F1 crosses between C57BL/6 (B6) and BALB/c (C) mice. Using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, we found that treatment with MC had no effect on the levels of GST enzyme activity in either the fetal lung or liver in either of the two parental strains or their F1 crosses. Low levels of expression of each of the four enzymes were detected by Western blotting in both fetal lung and liver tissues in all four strains. A statistically significant 3.5-fold induction was observed only for GSTμ in the fetal lung of the parental strain of BALB/c mice 48 h after exposure to MC. None of the other enzymes showed any significant differences in the levels of expression following exposure to MC. Although strain-specific differences in the expression of the GSTs that were independent of MC treatment were observed, they could not account for the differences previously observed in either the Ki- ras mutational spectrum or lung tumor incidence in the different strains of mice. Similar results were obtained when the maternal metabolism of MC was assayed in liver microsomal preparations. The results are consistent with previous studies showing low levels and poor inducibility of phase II enzymes during gestation, and demonstrate for the first time that all four of the major GST enzymes are expressed in fetal tissues. While the high inducibility of activating enzymes, such as Cyp1a1, and low, uninducible levels of phase II conjugating enzymes probably account for the high susceptibility of the fetus to transplacentally induced tumor formation, the results also suggest that factors other than metabolism may account for the strain-specific differences in susceptibility to carcinogen-mediated lung tumor induction following in utero exposure to chemical carcinogens.

Sulfur Amino Acid Restriction Induces the π Class of Glutathione S-Transferase Expression in Primary Rat Hepatocytes

The regulation of genes by amino acids is attracting increasing attention. In the present study, we investigated the restriction of expression of the pi class of glutathione S-transferase (GST Yp) by sulfur amino acids. Hepatocytes isolated from male Sprague-Dawley rats were cultured with L-15-based medium containing low (LSAA; 0.1 mmol/L L-methionine and 0.1 mmol/L L-cysteine) or high (HSAA; 0.5 mmol/L L-methionine and 0.2 mmol/L L-cysteine) amounts of sulfur amino acids for up to 6 d. Cellular protein contents did not differ between LSAA- and HSAA-treated cells over the entire period. In contrast, glutathione concentrations were suppressed by the LSAA medium and on d 6 were only 20% of those of HSAA-treated cells (P < 0.05). As shown by immunoblot analysis, GST Yp protein levels were greater in LSAA-treated cells than in HSAA-treated cells (P < 0.05). The induction of GST Yp by L-methionine and L-cysteine restriction was not affected by insulin and dexamethasone, but the latter suppressed GST Yp expression (P < 0.05). LSAA increased GST Yp mRNA levels and GST activity toward ethacrynic acid (P < 0.05). GST Yp induction occurred only in cells with a limited supply of L-methionine; restriction of L-isoleucine, L-leucine, L-lysine, and L-phenylalanine had no significant effect. In contrast with the induction of GST Yp, the expression of the GST isoforms Ya and Yb was not changed by amino acid restriction. In conclusion, hepatic GST Yp gene expression is upregulated by a limited availability of sulfur amino acids.

Detoxification of Herbicides in Phragmites australis

Unintentional loss of herbicides into drainage ditches, shores or other waterbodies may cause large problems in farmland. Therefore strategies for the phytoremediation of agrochemicals and especially herbicides have become a topic of great interest in many agricultural areas. However, in order to establish effective biological pollution control, information on the detoxification capacity of riparian plants and aquatic macrophytes (e.g., Phragmites australis) is important to build up effective buffer stripes. We determined the detoxification capacity of Phragmites australis roots and leaves for the conjugation of agrochemicals to glutathione by assaying the model substrate CDNB as well as the herbicides fenoxaprop-P, propachlor, pethoxamid and terbuthylazine. Specific GST activities were always higher in the rhizomes (6.78 +/- 0.88 microkat/mg protein for CDNB) than in leaves (1.08 +/- 0.21 microkat/mg protein). The detoxification capacity is distributed across an array of GST isoforms. In summary, Phragmites australis seems to be efficient in herbicide detoxification and a good candidate for phytoremediation of effluents from agricultural sites.

The interaction of selected natural products with human recombinant glutathione transferases

The interaction of geshoidin, diospyrin and ergothioneine, with heterologously expressed human glutathione transferases (GSTs) was investigated in vitro. Diospyrin and geshoidin inhibited the three GST isoforms tested, with IC50 values in the range 0.1-0.5 microm, whereas ergothioneine had no effect on the GSTs. The predominant mode of inhibition was noncompetitive with respect to both glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Diospyrin, however, competitively inhibited A1-1 and M1-1 with respect to GSH and geshoidin displayed mixed inhibition toward A1-1 with respect to GSH. The Ki values for diospyrin with respect to both GSH and CDNB were in the range 0.08-0.6 microM and those for geshoidin were in the range 16-173 microM. These results indicate that diospyrin is a potent inhibitor of heterologously expressed human GSTs A1-1, M1-1 and P1-1. Diospyrin and geshoidin were also found to inactivate P1-1 with diospyrin being a potent inactivator. Given these inhibitory properties, diospyrin may be a potential GST chemomodulator. Ergothioneine inactivated P1-1 only after preincubation and it enhanced ethacrynic acid inactivation of P1-1. Inactivation of P1-1 by ergothioneine may have implications for the antioxidant roles of P1-1 and ergothioneine in vivo.

Effect of three xenobiotic compounds on Glutathione S-Transferase in the clam Ruditapes decussatus

The effects of 4,4′DDE, methoxychlor and imidazole were studied on glutathione S-transferase activities in the gills and hepatopancreas of the clam Ruditapes decussatus. The contamination doses were 0.14 μM for 4,4′DDE, 0.14 μM for methoxychlor and 25 μM for imidazole. GST activities were spectrophotometrically measured. SDS-PAGE and isoelectric focusing (IEF) were used to separate the different GST isoforms in control and treated animals, followed by Western blotting performed with anti-alpha, anti-mu and anti-pi GST anti-sera. In the hepatopancreas, GST-CDNB activities were always two to five-fold lower than in the gills where the activities were significantly increased after exposure to 4,4′DDE (ca. 1.6-fold) and to methoxychlor (ca. 1.3-fold) compared to the controls (ca. 3 μmol min −1 mg −1protein) whereas they remained unchanged after treatment with imidazole. When using glutathione S-transferase anti-alpha, anti-mu and anti-pi anti-sera, a single 26 kDa polypeptide was observed in the hepatopancreas and in the gills in all the tested conditions. Five GST subunits were observed after IEF showing greater immuno-reactivity with the anti-pi mammalian class antiserum than with the anti-alpha or anti-mu mammalian anti-sera. One isoform of p I 5.77 was particularly induced by 4,4′DDE and methoxychlor; it was recognized by the three anti-sera tested and seemed to be more efficient in the gills than in the hepatopancreas. This isoform may play a role in organochlorine detoxication.

Association between glutathione S-transferase p1 polymorphisms and lung cancer risk in Caucasians: a case-control study

Glutathione transferases (GSTs), a multiple gene family of phase II enzymes, catalyze detoxifying endogenous reactions with glutathione and protect cellular macromolecules from damage caused by cytotoxic and carcinogenic agents. Glutathione S-transferase p1 (GSTP1), the most abundant GST isoform in the lung, metabolizes numerous carcinogenic compounds including benzo[ a]pyrene, a tobacco carcinogen. Previous studies suggest that genetic polymorphisms of GSTP1 exon 5 (Ile105Val) and exon 6 (Ala114Val) have functional effects on the GST gene product resulting in reduced enzyme activity. Individuals with reduced GST enzymatic activity may be at a greater risk for cancer due to decreased detoxification of carcinogenic and mutagenic compounds. Utilizing a hospital-based case-control study, we investigated the association between GSTP1 polymorphisms at exons 5 and 6 with lung cancer risk. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to successfully genotype the GSTP1 exons 5 and 6 polymorphism in 582 Caucasian lung cancer cases and 600 frequency matched Caucasian controls. There was no association between the exon 5 variant genotypes (A/G+G/G) and overall lung cancer risk (OR=1.09; 95% CI 0.82–1.45) nor when stratified by age, gender, and smoking status. However, the exon 6 variant genotypes (C/T+T/T) were associated with a statistically significant elevated lung cancer risk (OR=1.40; 95% CI 1.06–1.92). Additionally, there was an increase in lung cancer risk for the exon 6 variant genotypes in younger individuals (<62 years) (OR=1.63; 95% C.I. 1.07–2.49) but no effect in older individuals (OR=1.14; 95% CI 0.72–1.81). A statistically significant increased risk of lung cancer was also observed for the exon 6 variant genotypes among men (OR=2.17; 95% CI 1.41–3.33), but not among women (OR=0.80; 95% CI 0.51–1.28). Among ever smokers, the exon 6 variant genotypes were associated with an elevated lung cancer risk (OR=1.58; 95% CI 1.14–2.19), which was not evident for never smokers (OR=0.53; 95% CI 0.21–1.33). These data demonstrate that the GSTP1 exon 6 polymorphism, but not the exon 5 polymorphism, is associated with lung cancer risk that is especially evident in men, younger individuals, and ever smokers.

Protein expression profiling of glutathione S-transferase pi null mice as a strategy to identify potential markers of resistance to paracetamol-induced toxicity in the liver.

GST pi (GSTP) is a member of the glutathione S-transferase (EC 2.5.1.18; GST) family of enzymes that catalyse the conjugation of electrophilic species with reduced glutathione and thus play an important role in the detoxification of electrophilic metabolites. Deletion of GSTP in mice has previously been shown to lead to enhanced susceptibility to chemical-induced skin carcinoma, consistent with its known metabolic functions. A decreased susceptibility to paracetamol hepatotoxicity has also been observed, which has not been fully explained. One possibility is that deletion of the GSTP gene locus results in compensatory changes in other proteins involved in defence against chemical stress. We have therefore used complementary protein expression profiling techniques to perform a systematic comparison of the protein expression profiles of livers from GSTP null and wild-type mice. Analysis of liver proteins by two-dimensional electrophoresis confirmed the absence of GSTP in null mice whereas GSTP represented 3-5% of soluble protein in livers from wild-type animals. There was a high degree of quantitative and qualitative similarity in other liver proteins between GSTP null and wild-type mice. There was no evidence that the absence of GSTP in null animals resulted in enhanced expression of other GST isoforms in the null mice (GST alpha, 1.48%, GST mu, 1.68% of resolved proteins) compared with the wild-type animals (GST alpha, 1.50%, GST mu, 1.40%). In contrast, some members of the thiol specific antioxidant family of proteins, notably antioxidant protein 2 and thioredoxin peroxidases, were expressed at a higher level in the GSTP null mouse livers. These changes presumably reflect the recently described role of GSTP in cell signalling and may underlie the protection against paracetamol toxicity seen in these animals.

Pro-inflammatory Cytokines Tumor Necrosis Factor α and Interleukin-6 and Survival Factor Epidermal Growth Factor Positively Regulate the Murine GSTA4 Enzyme in Hepatocytes

We hypothesized that glutathione transferases could be induced and may participate to cellular defenses against the oxidative stress occurring during liver regeneration. Here, we evidenced that murine GSTA1 (mGSTA1), A4, Pi, and Mu are up-regulated during mouse liver regeneration, exhibiting a biphasic pattern of induction correlating early G(1) phase and G(1)/S transition of the cell cycle. Using confocal microscopy immunolocalization and subcellular fractionation, mGSTA4 was demonstrated in both mitochondria and cytosol and found preferentially increased in cytosol during liver regeneration. In addition, mGSTA4 was induced in vivo and in cultured hepatocytes by tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), and epidermal growth factor (EGF), factors that play crucial roles in hepatocyte survival and proliferation during liver regeneration. However, the mitogenic effect of EGF was not responsible for the induction of mGSTA4. In transient transfections, IL-6 and EGF, but not TNFalpha, transactivated the human GSTA4 (hGSTA4) promoter cloned upstream of the luciferase reporter gene suggesting that IL-6 and EGF up-regulated hGSTA4 at a transcriptional level, whereas TNFalpha could rather act at a post-transcriptional level. The inhibition of phosphoinositide 3-kinase, p38 MAPK, and MEK/ERK signaling pathways, using specific inhibitors, prevented EGF-dependent induction of mGSTA4 and transactivation of hGSTA4 promoter. Altogether, these data favor the conclusion that, in regenerating hepatocytes, several GST isoforms are induced and that cytokines TNFalpha and IL-6 and survival factor EGF positively regulate mGSTA4 via survival signaling pathways.

Differential effects of iron overload on GST isoform expression in mouse liver and kidney and correlation between GSTA4 induction and overproduction of free radicles

We have investigated the effect of iron overload on the expression of mouse GSTA1, A4, M1, and P1 in liver, the main iron storage site during iron overload, and in kidney. In iron-overloaded animals, mRNA and protein levels of GSTA1, A4, and M1 were increased in liver. In kidney, GSTA4 protein level was also increased while, unexpectedly, GSTA1 and M1 expression was strongly decreased. We showed, by immunohistochemistry, that GSTA4 was more abundant in hepatocytes of periportal areas and in convoluted proximal tubular cells in normal liver and kidney, respectively. In iron-overloaded mice, GSTA4 staining was more intense in cells that preferentially accumulated iron, and conjugation of 4-hydroxynonenal, a specific substrate of GSTA4, was enhanced in both organs. Moreover an acute exposure of primary cultures of mouse hepatocytes to iron-citrate strongly induced oxidative stress and cellular injury and resulted in an increase in GSTA4 expression, while cotreatment with iron-citrate and either desferrioxamine or vitamin E prevented both toxicity and GSTA4 induction. These data demonstrate that GSTA1 and M1 are differentially regulated in liver and kidney while GSTA4 is induced in both organs during iron overload. Moreover, they support the view that iron-induction of GSTA4 is related to an overproduction of free radicals.

A novel glutathione transferase from Haemophilus influenzae which has high affinity towards antibiotics

Cytosolic glutathione transferase (GSTs) are a family of multi-functional proteins which catalyse the conjugation of glutathione (GSH) to a large variety of endogenous and exogenous electrophilic compounds. Much is known about cytosolic mammalian GSTs, however, the presence of GSTs in several aerobic and anaerobic micro-organisms has also been demonstrated. Several findings seem to suggest that bacterial GSTs are involved in processes of biodegradation of xenobiotics, including antibiotics. However, the function played by these enzymes in the bacterial cell still remains to be clarified. At present, it is ill-defined whether bacterial GST can be classified, as in the case of mammalian enzymes, into several distinct classes. Here we report the purification of a GST isoform from Haemophilus influenzae using GSH-affinity chromatography. The purified protein was characterised by immunological and kinetic properties different from other known GSTs. The dissociation constants of chloramphenicol, ampicillin, rifampicin and tetracycline to the purified enzyme were 0.62, 9.06, 4.08 and 1.77 μM, respectively, as determined by following the quenching of the protein intrinsic fluorescence. These values were much lower than those previously determined for the same drugs with other mammalian or bacterial GSTs. The present results indicate that the enzyme purified from H. influenzae is a novel GST isoform well distinguished from other known mammalian or bacterial GSTs.

Phosphono analogs of glutathione: inhibition of glutathione transferases, metabolic stability, and uptake by cancer cells

Glutathione transferases (GSTs) have been shown to play an important role in multiple drug resistance in cancer chemotherapy. The inactivation of GST isoforms could lead to an enhanced activity of cytotoxic drugs. Thus, we have developed glutathione phosphono analogs [( S)-γ-glutamyl-(2 RS)-(±)-2-amino-(dialkoxyphosphinyl)-acetylglycines], which were previously shown to be inhibitors of GSTP1-1. In the present study, the inhibition characteristics of these analogs, including isoenzyme specificities, type of inhibition, and determination of K i values, were determined. The inhibition of class alpha GSTs was competitive towards GSH. A mixed-type, non-competitive inhibition of class mu and pi GSTs was observed. The K i values varied between 880 ± 210 and 0.45 ± 0.1 μM. The inhibitors were most effective towards class mu GSTs. In order to investigate the potential use of these GST inhibitors in intact cellular systems, two additional approaches were examined. Firstly, the metabolic stability was tested with purified γ-glutamyl transpeptidase and cell homogenates as well as during incubation of cell lines. No appreciable degradation was observed in any of the tested systems. Secondly, to facilitate cellular uptake, three derivatives were synthesized in which the glycine carboxylic group was esterified. Uptake and a possible intracellular cleavage to the corresponding free acids were monitored by HPLC analysis. The esters were effectively transported into HT29 (colon cancer) and EPG85-257P (gastric cancer) cells, respectively, and readily converted into the more active free acids. In conclusion, the tested inhibitors may be regarded as model compounds for the development of modulating agents in cancer chemotherapy.

Role of cytochrome P450 and glutathione S-transferase α in the metabolism and cytotoxicity of trichloroethylene in rat kidney

The toxicity and metabolism of trichloroethylene (TRI) were studied in renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. TRI was slightly toxic to both PT and DT cells, and inhibition of cytochrome P450 (P450; substrate, reduced-flavoprotein:oxygen oxidoreductase [RH-hydroxylating or -epoxidizing]; EC 1.14.14.1) increased TRI toxicity only in DT cells. In untreated cells, glutathione (GSH) conjugation of TRI to form S-(1,2-dichlorovinyl)glutathione (DCVG) was detected only in PT cells. Inhibition of P450 transiently increased DCVG formation in PT cells and resulted in detection of DCVG formation in DT cells. Formation of DCVG in PT cells was described by a two-component model (apparent V max values of 0.65 and 0.47 nmol/min per mg protein and K m values of 2.91 and 0.46 mM). Cytosol isolated from rat renal cortical, PT, and DT cells expressed high levels of GSH S-transferase (GST; RX:glutathione R-transferase; EC 2.5.1.18) α (GSTα) but not GSTπ. Low levels of GSTμ were detected in cortical and DT cells. Purified rat GSTα2–2 exhibited markedly higher affinity for TRI than did GSTα1–1 or GSTα1–2, but each isoform exhibited similar V max values. Triethyltinbromide (TETB) (9 μM) inhibited DCVG formation by purified GSTα1–1 and GSTα2–2, but not GSTα1–2. Bromosulfophthalein (BSP) (4 μM) only inhibited DCVG formation by GSTα2–2. TETB and BSP inhibited approximately 90% of DCVG formation in PT cytosol but had no effect in DT cytosol. This suggests that GSTα1–1 is the primary isoform in rat renal PT cells responsible for GSH conjugation of TRI. These data, for the first time, describe the metabolism of TRI by individual GST isoforms and suggest that DCVG feedback inhibits TRI metabolism by GSTs.

THE TOXICITY AND DETOXIFICATION OF α, β-UNSATURATED ALDEHYDES

trans-Cinnamaldehyde (CA) which is an α, β-unsaturated aldehyde, one of the most widely used flavoring agents in the world, is mutagenic in Salmonella, and is an inducer of the skin inflammation in rodents and in the human. In rat liver cytosol, the glutathione (GSH) conjugation of CA proceeded at a much higher rate than did its NADH-dependent reduction. CA was found to be a good substrate for the rat (r) liver class Mu GSH S-transferases (rGSTs) M2-2, M1-2, and MI-1 in decreasing order. The glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was irreversibly and highly 4(S)-selectively inactivated by the enantiomers of racemic 4-hydroxy2(E)-nonenal (4-HNE), a reactive product released from biomembranes by lipid peroxidation in cells. IC50 of 4(R)HNE for GAPDH was 3.6-fold higher than that of the 4(S)-enantiomer. In rat liver cytosol, the 4-HNE was detoxified highly 4(S)-selectively by GSH conjugation and 4(R)-selectively by NADH-dependent reduction mediated by alcohol dehydrogenase (ADH). In the cytosol, however, the GSH conjugation of 4(R)-HNE proceeded at a much higher rate than did its ADH-mediated reduction. The minor GST isoform, A4-4, in the rat liver played a major role in the cytosolic (S)-selective GSH conjugation. The catalytic efficiency, kcat/Km, of purified rGSTA4-4 was 4-fold higher for 4(S)-HNE than for 4(R)-HNE. Because of its much smaller Km than that of 4(R)-HNE, 4(S)-HNE was preferentially detoxified by rGSTA4-4 when racemic 4-HNE was used as a substrate.

Effects of lead on glutathione S-transferase expression in rat kidney: a dose-response study.

Glutathione S-transferases (GST, EC 2.5.1.18) are a family of phase II detoxification enzymes involved in the conjugation of glutathione to a highly diverse group of compounds. The purpose of this study was to evaluate the dose-response effects of lead acetate administration on the expression of rat kidney GST. Sprague-Dawley rats were injected with doses of lead acetate ranging from 0.11 to 114 mg/kg (0.3 to 300 mumol/kg) for three consecutive days and sacrificed 24 h later. Kidney GST activity, GST isoform HPLC profiles, blood lead analysis, and electron microscopy were performed. A dose of 1.1 mg/kg lead acetate resulted in a blood lead level of 26 micrograms/dl and produced a significant increase in GST activity which continued to increase with dose up to 38 mg/kg. Morphological changes were detected at 3.8 mg/kg and increasing severity of cellular damage paralleled dose, blood lead levels, and changes in body weight. Individual GST isoforms exhibited different thresholds and maxima; rGSTP1 and rGSTM1 had thresholds of 1.1 and 3.8 mg/kg, respectively, very similar rates of increase with dose, and a maximum yield that was 450% above control at a dose of 38 mg/kg for both enzymes. rGSTA1 and rGSTA3 showed similar thresholds (1.1 mg/kg) and maximal fold increase (275%) but varied in the relative response to each dose. These results indicate that renal GST increases occur at lead levels which are environmentally significant, that these changes precede cellular damage, and suggest that GST may serve as a tissue biomarker of lead exposure.

Detoxification of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells.

Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamster V79 cells. To evaluate the influence of sterical crowding in the substrate on the activity of GSTP-1, the study was extended to the strongly mutagenic fjord-region (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene. GSTP1-1,reduced preferentially the mutagenicity (studied at the hprt locus) of (+)-anti and (+)-syn-DEs of benzo[a]pyrene (by 66 and 67%) as compared with the corresponding (-)-anti- and (-)-syn-enantiomers (by 15 and 13%). These results are in line with previous studies on the enantioselectivity of purified GSTP1-1 towards the DE isomers of benzo[a]pyrene and benzo[c]phenanthrene showing that enantiomers with (R)-configuration at the benzylic oxiranyl carbon are better substrates than those with (S)-configuration. Interestingly, the (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene were efficiently detoxified by GSTP-1-1 in the constructed cell line (reduction of mutagenicity by 66 and 64%). This study demonstrates that differences in the caalytic activity seen for purified GST towards individual mutagens do not necessarily reflect the detoxification of DEs by the same enzyme in a living cell and provides further evidence that specific human GSTs play a role in the detoxification of DEs of PAHs.

The Role of GlutathioneS-Transferases as a Defense against Reactive Electrophiles in the Blood Vessel Wall

The glutathione transferases (GSTs) are a family of ubiquitous enzymes that catalyze the conjugation of reduced glutathione (GSH) with reactive electrophiles. Rat vascular tissue contains GST isoforms that represent a major cellular defense mechanism against atherogenic α,β-unsaturated aldehydes (Misraet al., Toxicol. Appl. Pharmacol.133, 27–33, 1995). In this study we examined the role of GSTs in providing protection to cultured neonatal vascular smooth muscle cells (VSMCs) from the α,β-unsaturated carbonyl cardiovascular toxins, allylamine and its metabolite, acrolein. Confluent cultured cells were exposed to 2 to 10 μM allylamine (a cardiovascular toxin that is metabolizedin vivoandin vitroby VSMCs to the reactive aldehyde, acrolein) or to acrolein (2–10 μM) for 48 h; dose–cytotoxicity curves were generated utilizing a tetrazolium-dependent cytotoxicity assay. Concommittant treatment with sulfasalazine, an established inhibitor of GST, was found to markedly increase allylamine- or acrolein-induced cytotoxicity, decreasing the LC50by two- to threefold at 50 to 100 μM sulfasalazine. A clonogenic survival assay in VSMCs exposed to these compounds for 4 h confirmed lethal toxicity and enhanced toxicity following cotreatment with sulfasalazine. Isobologram analysis (which statistically defines the limits of additivity of two independent treatments) showed that the sulfasalazine effect on both allylamine and acrolein cytotoxicity was supraadditive, or synergistic. Sulfasalazine was not cytotoxic to VSMCs in the range of concentrations that augmented acrolein or allylamine cytoxicity; total GST activity was inhibited, however, in a dose-dependent manner in that range. GST purified by GSH-affinity chromatography from pelleted untreated cells gave specific activities and kinetic constants consistent with those previously reported for rat aorta total GSTs. The catalytic efficiency (Kcat/Vm) was found to be much greater for 4-hydroxy-2-nonenal than for 1-chloro-2,4-dinitrobenzene (0.058 vs 0.4 s-1mM-1). Western blot of purified total GSTs using antibodies against rec-mGSTA4–4 revealed a single band at 25 kDa, confirming the presence of a GST isozyme immunologically similar to rat GST8–8, which is known to utilize α,β-unsaturated carbonyls as preferred substrates. Our data indicate that GSTs are an important defense in the vascular media, protecting blood vessels against α,β-unsaturated carbonyl cardiovascular toxins that are involved in initiating atherosclerotic lesions.

Polymorphic expression of biotransformation enzymes in Barrett's oesophagus and oesophageal carcinomas

Background: Susceptibility to oesophageal cancer or Barrett's epithelium, with 30-125 fold increase for development of adenocarcinoma, of a particular individual may depend in part on the balance between phase I drug metabolizing (e.g. cytochrome P450 IA1; CYPIA1) and phase II detoxification enzymes (e.g. glutathione S-transferase; GST). Polymorphic variants in exon 7 (CYPIAIlNcol) and 3' flanking region (CYPIAIlMspl) of the CYPIA1 gene may lead to increased levels of bioactive compounds. In addition GSTM1 and GSTF1 null genotypes, resulting in complete absence of these GST isoforms and consequently resulting in deficient detoxification, were described. Aims: To study the frequency of polymorphic variants in the CYPIA1 gene in combination with GSTM1 and GSTF1 null genotypes in patients with squamous cell carcinoma (SCC, n=ll), adenocarcinoma (AC, n=23) or Barrett's epithelium (BE, n=56) in the oesophagus as compared to healthy blood donors (BD, n=207). Methods: DNA was extracted from whole blood and genetic polymorphisms were studied by polymerase chain reaction (PCR) based techniques. Chi square analysis was used for statistical evaluation. Results: CYPIAI/Ncol as well as CYPIAIlMspl rare alleles were more common in patients with SCC (each 38.5%) as compared to BD (Ncol 23.5%, Z2=4.07, p=0.04; Mspl 15.9%, ;~2=4.34, p=0.04). No differences in CYPIA1 polymorphic variants were found between the other populations. GSTM1 and GSTT1 null genotypes did not differ between the patient and control populations. Summary/conclusions: Presence of rare alleles in CYPIA1 exon 7 or the 3' flanking region, both leading to increased bioactivation of precarcinogens, may result in increased susceptibility to squamous cell carcinoma of the oesophagus.

Studies on the Relationship between Estrogen Receptor Content, GlutathioneS-Transferase π Expression, and Induction by 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Drug Resistance in Human Breast Cancer Cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces both phase I and phase II drug-metabolizing enzymes in rodent liver and hepatoma cell lines and this induction is mediated by the aryl hydrocarbon (Ah) receptor. Induction of CYP1A1 by TCDD in human breast cancer cells has been reported and results of several studies suggest that the estrogen receptor (ER) may be required for Ah responsiveness. This study investigates the induction of GSTπ by TCDD in human breast cancer cells and the role of the ER in mediating this response. TCDD did not induce chloramphenicol acetyl transferase (CAT) activity in ER positive (ER+) MCF-7 and ER−MDA-MB-468 and MDA-MB-231 human breast cancer cell lines transiently transfected with GSTπ (human) or GSTP (rat) promoter–reporter constructs containing the −291/+36 and −2.9/+59 region, respectively, of the GSTπ and GSTP gene promoters. Furthermore, TCDD did not induce GSTπ or GSTP in MDA-MB-468 and MDA-MB-231 human breast cancer cells stably transfected with the ER. RT-PCR confirmed that GSTπ mRNA levels were low in ER+MCF-7 cells and high in ER−MDA-MB-468 and MDA-MB-231 cells; however, in MDA-MB-468 and MDA-MB-231 cells stably transfected with the ER GSTπ mRNA levels remained elevated and were not inducible. MDA-MB-468 and MDA-MB-231 cells stably transfected with the ER exhibited increased GST activity and decreased GSH content compared to wild-type cells; however, in MDA-MB-468 cells stably transfected with ER, the susceptibility to doxorubicin, ellipticine, chlorambucil, malphalan, or cisplatin was similar to that observed in wild-type cells. Adriamycin accumulation was similar in wild-type and ER stably transfected cells and verapamil did not affect this response, suggesting that ER expression did not influence p-glycoprotein activity. Taken together these data suggest that not all GST isoforms are responsive to TCDD and stable transfection of ER−cells with ER is not sufficient to restore the ER+phenotype in some breast cancer cell lines.